The present invention relates generally to fluorescence imaging systems and, more particularly, to a method and apparatus for fluorescence imaging in which the emission detection wavelength is continuously tunable.
In the biotechnical field, fluorescent dyes are routinely used as sensitive, non-isotopic labels. These labels are used to identify and locate a variety of cell structures such as specific chromosomes within a DNA sequence. One application, fluorescence in situ hybridization (FISH), first attaches specific chromosome regions with DNA probes and then images the probes using microscopy.
In a paper by Thomas Ried et al. entitled "Simultaneous Visualization of Seven Different DNA Probes by In Situ Hybridization Using Combinatorial Fluorescence and Digital Imaging Microscopy," Proc. Natl. Acad. Sci. USA:Genetics, 89 (February 1992), the authors describe a combinatorial probe labeling scheme. The disclosed technique increases the number of target sequences that can be simultaneously detected using a given number of fluorochromes. Specifically, the authors disclose simultaneously analyzing up to seven probes using only three fluorochromes.
A variety of devices have been designed to read fluorescent-labeled samples. In general, a device designed to read and/or image a fluorescent-labeled sample requires at least one light source emitting at one or more excitation wavelengths and means for detecting one or more fluorescent wavelengths.
In U.S. Pat. No. 5,290,419, a multi-color fluorescence analyzer is described which irradiates a sample with two or more excitation sources operating on a time-shared basis. Band pass filters, image splitting prisms, band cutoff filters, wavelength dispersion prisms and dichroic mirrors are use to selectively detect specific emission wavelengths.
In U.S. Pat. No. 5,213,673, a multi-colored electrophoresis pattern reading apparatus is described which irradiates a sample with one or more light sources. The light sources can either be used individually or combined into a single source. Optical filters are used to separate the fluorescence resulting from the irradiation of the sample into a plurality of fluorescence wavelengths.
In U.S. Pat. No. 5,190,632, a multi-colored electrophoresis pattern reading apparatus is described in which one or more light sources are used to generate a mixture of light capable of exciting two or more fluorescent substances. Both optical filters and diffraction gratings are used to separate the fluorescence by wavelength.
In U.S. Pat. No. 5,062,942, a fluorescence detection apparatus is described in which a fluorescent light image is separated into a plurality of virtual images. Bandpass filters are used to separate the virtual images by wavelength.
In an article by Cothren et al. entitled "Gastrointestinal Tissue Diagnosis by Laser-Induced Fluorescence Spectroscopy at Endoscopy," Gastrointestinal Endoscopy 36 (2) (1990) 105-111, the authors describe an endoscopic system which is used to study autofluorescence from living tissue. The excitation source is monochromatic with a wavelength of 370 nanometers. Optical fibers are used to collect the fluorescence emitted by the irradiated tissue. Emission spectra are collected from 350 to 700 nanometers using an imaging spectrograph coupled to a gated optical multi-channel analyzer. A similar autofluorescence system was described by Andersson et al. in "Autofluorescence of Various Rodent Tissues and Human Skin Tumour Samples," Lasers in Medical Science 2 (41) (1987) 41-49.
The above fluorescence analyzers suffer from a number of performance disadvantages. For example, all of the systems have a very limited selection of excitation wavelengths; none of them give the user the ability to specify any particular excitation wavelength. Thus these systems do not allow the user to optimize the excitation of the fluorescent label. Furthermore, the prior art systems generally detect fluorescence in discrete wavelength bands as opposed to being continuously tunable over the detection wavelengths of interest. Without the ability to continuously tune the excitation and emission detection wavelengths, the user is not able to peak the fluorescent response.
The lack of continuous tunability of the detection wavelengths in the prior art fluorescence analyzers is especially problematic in those instances in which the chosen fluorescent labels undergo spectral shifts due to external environmental effects. For example, some fluorescent probes exhibit sensitivity to solvent polarity, solvent in this context including the interior regions of various biomolecular structures (e.g., cells, membranes, proteins, etc.). This phenomena is commonly observed in fluorescent probes which have large excited-state dipole moments. Another commonly observed cause of fluorescence spectral shifts is the pH sensitivity of many fluorescent labels. Generally, pH sensitivity is the result of a reconfiguration in the probe's .pi.-electron system.
From the foregoing, it is apparent that a fluorescence analyzer is desired which is continuously tunable over the emission detection wavelengths.